The present invention is in the field of the intracellular delivery of therapeutic agents, and more particularly in the area of enhancement of transport or delivery of molecules into the cell cytosol, using bispecific affinity reagents and pH-responsive, membrane destabilizing polymers.
It is often difficult to deliver compounds, such as proteins, genetic material, and other drugs and diagnostic compounds, intracellularly because cell membranes resist the passage of these compounds. Various methods have been developed to administer agents intracellularly. For example, genetic material has been administered into cells in vivo, in vitro and ex vivo using viral vectors, DNA/lipid complexes and liposomes. DNA has also been delivered by synthetic cationic polymers and copolymers and natural cationic carriers such as chitosan. Sometimes the synthetic polymers are hydrophobically modified to enhance endocytosis. While viral vectors are efficient, questions remain regarding the safety of a live vector and the development of an immune response following repeated administration. Lipid complexes and liposomes appear less effective at transfecting DNA into the nucleus of the cell and may potentially be destroyed by macrophages in vivo.
Receptor mediated endocytosis offers an alternative means to target specific cell types and to deliver therapeutic agents intracellularly. Receptor-mediated endocytosis (RME) occurs when ligands bind to cell surface receptors on eukaryotic cell membranes, initiating or accompanying a cascade of phenomena culminating in the cellular invagination of membrane complexes within clathrin-coated vesicles. Compounds which interact with specific cell surface receptors are employed to target specific cell surface receptors. The compounds are endocytosed into the endosomes once the compounds interact with the cell surface receptors. Linkages have been made directly with the compounds, or, in the case of DNA, through conjugation with polycationic polymers such as polylysine and DEAE-dextran which are then complexed with the DNA. (Haensler et al., Bioconj. Chem., 4:372-379 (1993)).
Even after therapeutic agents are delivered intracellularly, normal trafficking in the cell can minimize their effectiveness. For example, certain antibody-antigen conjugates are readily endocytosed. However, after endocytosis, the antibody is not released into the cytosol but rather remains isolated in endosomes until it is trafficked to a lysosome for degradation. (Press, O. W. et al., Cancer Research, 48: 2249-2257 (1988)). Endosomes are membrane bound phospholipid vesicles which function in intracellular trafficking and degradation of internalized proteins. The internal pH of the endosomes is between 5.0 and 5.5. Genetic material, being negatively charged, is often complexed with polycationic materials, such as chitosan and polylysine, for delivery to a cell. Both immunotherapy and gene therapy using polycation/nucleic acid complexes are limited by trafficking of the complexes by the cell from endosomes to lysosomes, where the antibody conjugates or nucleic acids are degraded and rendered ineffective.
Protein transduction domains (PTDs) have attracted considerable interest in the drug delivery field for their ability to translocate across biological membranes. The PTDs are relatively short (11-35 amino acid) sequences that confer this apparent translocation activity to proteins and other macromolecular cargo to which they are conjugated, complexed or fused (Derossi et al., 1994; Fawell et al., 1994; Elliott and O'Hare, 1997; Schwarze et al., 2000; Snyder and Dowdy, 2001; Bennett et al., 2002).
The highly cationic 11 amino acid residue (YGRKKRRQRRR) PTD from the human immunodeficiency virus (HIV-1) TAT protein (Frankel and Pabo, 1988; Green and Loewenstein, 1988) has been one of the most well-studied translocating peptides. In-frame fusion proteins containing the TAT sequence were shown to direct cellular uptake of proteins that retained their activity intracellularly (Nagahara et al., 1998; Kwon et al., 2000; Becker-Hapak et al., 2001; Jo et al., 2001; Xia et al., 2001; Cao et al., 2002; Joshi et al., 2002; Kabouridis et al., 2002; Peitz et al., 2002). Subsequently, a diverse collection of over 60 full-length proteins with functional domains from 15 to 120 kDa have been engineered to date. Various studies employing TAT-fusion methodologies have demonstrated transduction in a variety of both primary and transformed mammalian and human cell types, including peripheral blood lymphocytes, diploid fibroblasts, keratinocytes, bone marrow stem cells, osteoclasts, HeLa cells and Jurkat T-cells (Fawell et al., 1994; Nagahara et al., 1998; Gius et al., 1999; Vocero-Akbani et al., 1999, 2000, 2001; Becker-Hapak et al., 2001). Furthermore, in vivo intracellular delivery by injection of a TAT-b-gal fusion has been demonstrated (Schwarze et al., 1999; Barka et al., 2000). However, intracellular delivery by TAT and other peptide domains is inefficient, irreproducible and in many cases results have been misleading due to artifacts caused by fixation procedures (Richard et al., J. Biol. Chem. 278:585, 2003).
In addition to drug delivery, there are many potential in vitro applications in areas such as drug discovery and laboratory assays that could benefit from improved intracellular delivery of biomolecules and macromolecular cargo. However, certain challenges remain. For example, even if the biomolecules and macromolecular cargo can be targeted to the desired cells and endocytosed by the cells, often are not effectively released from endosomes into the cytosol, but are degraded by lysosomes. These and other challenges are addressed by embodiments of the present invention.